C DAB

day 2 day 4

Φ (fmol/min./oocyte)5-HT //

0 0.1 1 10 100

0 25 50 75 100

NormalizedΦ 5-HT //

0 0.1 1 10 100

0 10 20 30 40

//

cRNA injected (ng/oocyte) I (5-HT) (- nA)

0 0.1 1 10 100

0 25 50 75 100 day 2

day 4

//

cRNA injected (ng/oocyte) Normalized I (5-HT)

Figure 11. Increasing culture time decreases cRNA potency for Φ5-HT but not I5-HT.

Oocytes are injected with hSERT cRNA and cultured for 2 days (filled symbols, dashed lines) or 4 days (open symbols, solid lines) at 24ºC. A, Φ5-HT measured in 2.5 min. assays. B, Φ5-HT normalized to the fitted maximum from A. Lines represent fits to the Hill equation (day 2, Φ5-HTmax = 1.5 ± 0.1 fmol/min./oocyte, EC50 cRNA = 0.8 ± 0.1 ng/oocyte, nH = 1.3; day 4, Φ_5-HTmax = 2.6 ± 0.1

fmol/min./oocyte, EC50 cRNA = 2.5 ± 0.1 ng/oocyte, nH = 1.3). C, I5-HT measured at –80 mV (note inverted axis). D, I5-HT normalized to the fitted maximum from C. Data represent means ± SEM from n = 4 – 6 oocytes/condition in a single representative experiment. Lines represent fits to the Hill equation (day 2, I5-HTmax= -28.7 ± 0.5 nA, EC50 cRNA = 1.5 ± 0.1 ng/oocyte, nH = 1.4 ± 0.1; day 4, I5- HTmax = -35.3 ± 0.5 nA, EC50 cRNA = 1.1 ± 0.1 ng/oocyte, nH = 1.3 ± 0.1).

In order to correlate hSERT function with surface expression, we treat cRNA- injected oocytes with a membrane-impermeant biotinylating reagent (Sulfo-NHS- biotin), solubilize oocytes, and isolate plasma membrane proteins with streptavidin

35

beads. Following SDS-PAGE, we visualize hSERT protein using a monoclonal anti- SERT antibody (ST 51-2). Fig. 12 shows a representative Western blot from hSERT cRNA-injected oocytes. hSERT immunoreactivity is absent in non-injected oocytes (lane 1, 2), indicating that ST 51-2 detects authentic hSERT protein. In lanes loaded with

protein from cRNA-injected oocytes, several forms of hSERT are detected: a small (~60 kDa) band that is present only in the intracellular fraction, a broad band (70-100 kDa) that is typical of mature hSERT ¹⁴², and a larger (~220 kDa) band in both surface and intracellular fractions that may represent an SDS-resistant SERT protein complex (Fig.

12). Band densities in biotinylated surface (S) and non-biotinylated intracellular (IC) fractions are comparable (Fig. 12, lanes 5, 6 and 7, 8) despite loading of IC lanes with 5%

of the total extract and S lanes with the biotinylated bead eluate. Surface SERT therefore represents no more than 5% of the total SERT protein in the oocyte.

biotin S IC S IC S IC S IC

cRNA (ng) 0 0.42 4.2 21

217123

71 48

Lane: 1 2 3 4 5 6 7 8

kDa

Figure 12. Surface hSERT expression depends on the amount of cRNA injected.

Oocytes are injected with SERT cRNA (0.41 to 41.4 ng/oocyte) and cultured for 2 days (24°C).

A representative Western blot showing hSERT protein in biotinylated (S, surface) or non- biotinylated (IC, intracellular) fractions is shown. Lanes represent protein from 20 oocytes/condition. Equal protein is loaded in each lane.

We also measure SERT function in the same batches of oocytes used for Western 36

0 0.1 1 10 100 0.00

0.25 0.50 0.75 1.00

////

surface SERT

I

_5-HT

Φ

_5-HT

cRNA injected (ng/oocyte)

Norm aliz ed Res pons e

Figure 13. Φ5-HT and I5-HT are differentially sensitive to hSERT expression level.

Oocytes are injected with SERT cRNA (0.41 to 41.4 ng/oocyte) and cultured for 2 days (24°C) Φ_5-HT (filled circles, dotted line) is measured in 2.5 min. (n = 3) or 30 min. (n = 2) assays. I_(5-HT) (open squares, solid line) is measured at -80 mV, pH 7.6. Surface SERT (filled triangles, dashed line) is measured by densitometry of mature surface SERT bands in Western blots. Data are expressed as a fraction of the indicated response in oocytes injected with 21 ng cRNA. Data represent means ± SEM (where shown) from n = 5 (Φ_5-HT), 4 (I(5-HT)), or 2 (surface SERT) separate experiments. Lines represent fits to the Hill equation (-HT, EC50 cRNA = 0.13 ± 0.03 ng/oocyte; I(5- HT), EC50 cRNA = 3.42 ± 0.86 ng/oocyte; surface SERT, EC_{50 cRNA} = 6.36 ± 0.81 ng/oocyte; n_H = 1.0 for all fits).

blotting. The relative densities of the mature surface hSERT (70-100 kDa) bands from two different oocyte batches are averaged and plotted against the amount of cRNA in Fig. 13. Hill fits to the data show that the cRNA-dependence of surface SERT, Φ5-HT, and I(5-HT) are not equal. As observed previously (Fig. 11), the cRNA-dependence of I(5- HT) is shifted rightward with respect to Φ5-HT (2 day culture, 24°C). At cRNA levels where Φ5-HTapproaches saturation (e.g. 1.5 ng/oocyte), I(5-HT) is still rising and represents only 35% of the current measured after injection of 21 ng/oocyte.

37

Surprisingly, the cRNA dependence of cell surface hSERT protein is similar to I(5-HT) and quantitatively different from Φ5-HT (Fig. 13, Φ5-HT, EC50 cRNA = 0.13 ± 0.03 ng/oocyte; I(5- HT), EC50 cRNA = 3.42 ± 0.86 ng/oocyte; surface SERT, EC50 cRNA = 6.36 ± 0.81 ng/oocyte;

nH = 1.0 for all fits). SERT protein correlates linearly with I(5-HT) (R² = 0.90), but not Φ5- HT, indicating that plasma membrane expression of SERT is selectively reported by I(5- HT).

A similar difference in cRNA potency is seen for rSERT in oocytes cultured for

0 5 10 15 20 25

0 50 100 150

Φ

5-HT

( fm ol/60 m in./oocyte)

I

_(5-HT)

(- nA)

Figure 14. Increasing SERT expression reveals a functional conversion from Φ5-HT mode to I5-HT mode.

Oocytes are injected with increasing amounts of rSERT cRNA and cultured for 14-16 days (18ºC). Φ_5-HT is measured in 60 min. assays (12 nM [³H]-5-HT) and I5-HT is at –80 mV, pH 7.6. Data represent means± SEM from n = 4 - 6 oocytes/condition in a single representative experiment. The solid line represents a fit to the exponential function y = a – bc^x, where a = 127.9 ± 15.3, b = 104.2 ± 20.9 and c = 0.53 ± 0.17. Prior to being plotted parametrically, data are fit to the Hill equation (Φ5-HTmax = 126.0 ± 3.1 fmol 5HT/60 min./oocyte, EC50 cRNA = 0.59 ± 0.02 ng/oocyte, n_H = 2.4 ± 0.2; I_(5-HT)max = -40.0 ± 4.4 nA, EC₅₀

cRNA = 20.0 ± 4.8 ng/oocyte, n_H = 1.0 ± 0.2).

38

14-16 days at 18°C to maximize expression (Fig. 14). In order to determine whether accumulation of significant 5-HT inside the oocyte affects the cRNA dependence of Φ5- HT and I(5-HT), we also increase incubation time for Φ5-HT assays to 60 min. Even under conditions of high SERT expression and increased absolute 5-HT accumulation (~125 fmol/oocyte), rSERT cRNA exhibits differential potency for Φ5-HT and I(5-HT). As for hSERT, increasing rSERT cRNA leads to sigmoidal rises in Φ5-HT and I(5-HT) that are evident when the data are plotted as in Fig. 13 (not shown). At 4.1 ng/oocyte rSERT cRNA, Φ5-HT has reached a plateau, but I(5-HT) increases 3.8-fold between 4.1 ng and 41.4 ng/oocyte cRNA (-7.1 ± 0.9 nA and -27.4 ± 2.6 nA, respectively, Fig. 14 data). Fig. 14 shows a parametric plot the data to emphasize the relationship between Φ5-HT and I(5- HT). In the low cRNA range (≤ 1 ng/oocyte), increases in cRNA lead predominantly to more Φ5-HT. An inflection point is reached at 2-5 ng/oocyte; above 2 ng/oocyte, increasing cRNA generates primarily additional I(5-HT) (Fig. 14). A simple model of SERT function predicts that the slope of the parametric plot will be independent of the number of SERT proteins (N, Eqn. 3), and thus reflects only intrinsic properties of SERT function (i, po, ν, q). However, the slope of the line fit to the data is not constant (Fig.

14), suggesting that cooperative interactions govern the relative magnitudes of Φ5-HT

and I(5-HT).

Fig. 15 shows a current trace from a representative oocyte injected with rSERT cRNA (14 ng). I(5-HT) is elicited first at pH 7.6, and subsequently at pH 5.0. After establishing a stable baseline current (defined as zero current by offline leak

subtraction), the oocyte is superfused with 5-HT (3.2 µM) long enough to determine steady-state 5-HT-induced current amplitude (Fig. 15, I(5-HT) pH 7.6), then returned to control solution. Superfusion of frog Ringer’s, pH 5.0, elicits a steady-state constitutive inward leak current (Fig. 15, IH+ leak) in both non-injected and cRNA-injected oocytes, but the magnitude of IH+ leak is larger in oocytes expressing SERT ^154,155. Superfusion of 5-HT (3.2 µM) at pH 5.0 potentiates 5-HT-induced current, (Fig. 15, I(5-HT) pH 5.0). H⁺- potentiation of I(5-HT) confirms rSERT expression (I(5-HT) in hSERT is insensitive to

39

extracellular acidification ¹⁵⁴) and allows us to more easily measure I(5-HT) in oocytes injected with small quantities of cRNA.

0 30 60 90 120

-50 -40 -30 -20 -10 0

I_H⁺ _leak = [I_pH5.0] - [I_pH7.6]

I(5-HT) pH 5.0 = [I5-HT, 3.2 µM, pH 5.0] - [I_{pH 5.0}] I(5-HT) pH 7.6 =

[I5-HT, 3.2 µM, pH 7.6] - [I_Control]

pH 5.0

I (n A )

Time (sec)

Figure 15. H⁺-potentiation of I(5-HT) in rSERT.

Oocytes are injected with 14 ng rSERT cRNA and cultured for 13 days (18°C). Data represent baseline-subtracted current from a single representative oocyte. Dashed line indicates the zero current level. Filled bars indicate time during which 5-HT (3.2 µM) is superfused and open bar indicates time during which frog Ringer’s, pH 5.0 is superfused.

Artifact spikes in current recording indicate actual time that superfusion switches are initiated; the delay between switch artifact and change in current represents the

combination of a) lag time for superfusion to reach the oocyte in the recording chamber, and b) penetration of 5-HT through the oocyte’s protective vitelline layer.

40

0 10 20 30 40 0

10 20 30 40

**

* *

transporter-like

cRNA injected (ng/oocyte)

channel-like

ρ

Figure 16. Increasing rSERT expression alters ρ.

Οocytes are injected with increasing amounts of rSERT cRNA and cultured for 14-16 days (18°C). I(5-HT) is measured at -80 mV, pH 5.0 during a 2 min. superfusion of 5-HT (3.2 µM) + [³H]-5-HT (30 nM). Q5-HT and Q(5-HT) are calculated as described to obtain ρ. Data represent means ± SEM from n = 3–6 oocytes/condition in a single representative experiment. Line represents a fit to the Hill equation (ρ_max = 37.5 ± 5.6 e/5-HT; EC50 cRNA = 10.2 ± 3.7

ng/oocyte; nH = 1.0). Statistically significant differences are indicated by *, p , 0.05, **, p <

0.01 versus oocytes injected with 1.4 ng cRNA.

In order to test whether the differential cRNA dependence of Φ5-HTand I(5-HT) is due to voltage differences intrinsic to the respective assay conditions, we measure Φ5- HTand I(5-HT) simultaneously under voltage clamp at –80 mV. Superfusion of 5-HT (3.2 µM) + [³H]-5-HT (30 nM) for 2 min. at 24°C elicits both Φ5-HT and I(5-HT) , from which we calculate charge movement associated with 5-HT flux (Q5-HT) and total charge

movement (Q(5-HT)) (Eqn. 2). Fig. 16 shows that ρ increases 4.3-fold as the amount of cRNA increases from 1.4 ng to 33.6 ng (ρ = 6.7 ± 1.6 and 28.6 ± 3.1, respectively). In contrast to I(5-HT), Φ5-HTis relatively insensitive to pH change ^77,154. We therefore

calculate that under physiological conditions (–80 mV, pH 7.6), ρ varies between ~1 and

41

~4 over a range of rSERT expression in oocytes. The validity of this calculation is reinforced by the observation that ρ = 4.4 when measured at pH 7.6 in a separate batch of oocytes injected with 8.3 ng/oocyte rSERT cRNA (Fig. 22). The differential cRNA dependence of I(5-HT) and Φ5-HT is therefore not due to differences in membrane

potential. We conclude that one or more microscopic properties of SERT function (Eqn.

3) are therefore sensitive to expression level.

Fig. 17 shows the H⁺-potentiation ratio of I(5-HT) in the same batch of oocytes as those shown in Fig. 16. I(5-HT) pH 7.6 and I(5-HT) pH 5.0 are highly correlated across the range of cRNA tested (R² = 0.99, Fig. 14 and data not shown), indicating that cRNA potency is

0 10 20 30 40

0 5 10 15

cRNA injected (ng/oocyte) I

(5-HT) pH 5.0

/ I

(5-HT) pH 7.6

Figure 17. H⁺-potentiation of I(5-HT) is independent of rSERT expression level.

Data is from oocytes shown in Fig. 16. I(5-HT) is measured at -80 mV, pH 7.6 and pH 5.0. Data represent means ± SEM from n = 4 - 10

oocytes/condition in a single representative experiment. Dashed line indicates average level of H⁺ potentiation across all cRNA injections tested.

42

0 1 10 100 0

25 50 75 100

0.1 µM 5-HT 1 µM 5-HT 10 µM 5-HT //

//

cRNA injected (ng/oocyte) Φ 5-HT (% Max.)

0 1 10 100

0 25 50 75 100

A B

1 µM 5-HT 3.2 µM 5-HT 32 µM 5-HT //

cRNA injected (ng/oocyte) I (5-HT) (% Max.)

Figure 18. Increasing 5-HT concentration decreases cRNA potency for Φ5-HT but not Ι5-HT. Oocytes are injected with increasing amounts of hSERT cRNA and cultured for 2 days (24°C). Φ5- HT is measured in the presence of 0.1 µM 5-HT (filled squares, dotted line), 1.0 µM 5-HT (open circles, sold line), or 10 µM 5-HT (filled triangles, dashed line) and normalized to the respective Φ_5-

HTmax. Lines represent fits to the Hill equation (0.1 µM 5-HT, EC50 cRNA = 0.45 ± 0.01 ng/oocyte; 1.0 µM 5-HT, EC_{50 cRNA} = 0.84 ± 0.04 ng/oocyte; 10 µM 5-HT, EC_{50 cRNA} = 2.20 ± 0.12 ng/oocyte; nH = 1.3). [³H]-5-HT concentration in all experiments is 30 nM and indicated 5-HT concentration is achieved by addition of non-labeled 5-HT. B, Ι_5-HT measured at different 5-HT concentrations (1.0 µM 5-HT (open circles, dashed line), 3.2 µM 5-HT (solid triangles, sold line), or 32 µM 5-HT (open squares, dotted line) and normalized to the respective Ι_5-HTmax. Lines represent fits to the Hill equation (1.0 µM 5-HT, EC50 cRNA = 3.0 ± 0.4 ng/oocyte; 3.2 µM 5-HT, EC50 cRNA = 3.1 ± 0.4

ng/oocyte; 32 µM 5-HT, EC_{50 cRNA} = 3.5 ± 0.5 ng/oocyte; nH = 1.0 for all fits). Data are expressed as a percentage of the maximal Φ_5-HT (A) or Ι_5-HT (B) measured for each condition. For A and B, data represent mean ± SEM from n = 4 - 6 oocytes/condition in a single representative experiment.

not altered by extracellular acidification. When measured directly, the ratio of I(5-HT) pH 7.6 to I(5-HT) pH 5.0 is independent of SERT expression level (Fig. 17, dashed line, I(5-HT) pH5.0/I(5-HT) pH7.6 = 8.8 ± 0.4, from 1.4 to 33.6 ng/oocyte). In contrast to the ratio of I(5-HT)

to Φ5-HT (Figs. 13, 14, 16), H⁺-potentiation conforms to the predicted behavior for SERTs operating according to a simple model (Eqn. 3).

43

10^-8 10^-7 10^-6 10^-5 10^-4 0

25 50 75 100

B A

Specific Φ5-HT (% Control)

[5-HT], (M)

10^-7 10^-6 10^-5 10^-4 10^-3 0

25 50 75 100

Specific Φ5-HT (% Control)

[cocaine], (M)

Figure 19. Potency for inhibition of Φ5-HT is sensitive to hSERT expression level.

Oocytes are injected with 0.42 ng/oocyte (open symbols, solid lines) or 21 ng/oocyte (filled symbols, dashed lines) hSERT cRNA and cultured for two days (24°C). A, Φ_5-HT is measured in the presence of increasing concentrations of non-labeled 5-HT (squares). Lines represent fits to the Hill equation (0.42 ng/oocyte, solid line, IC50 = 1.6 ± 0.3 µM; 21 ng/oocyte, dashed line, IC50 = 7.7 ± 0.9 µM; nH = 1.0). B, Φ5-HT is measured in the presence of increasing concentrations of cocaine (triangles). Lines represent fits to the Hill equation (0.42 ng/oocyte, solid line, IC50 = 1.9 ± 0.42 µM; 21 ng/oocyte, dashed line, IC50 = 20 ± 3.2 µM; n_H = 1.0). Data represent means ± SEM from n = 4 - 6

oocytes/condition in a single representative experiment.

In order to investigate whether other SERT properties are sensitive to SERT expression level, we examine hSERT pharmacology in oocytes injected with varying quantities of hSERT cRNA. As expected, Φ5-HT increases as 5-HT concentration

increases (0.1 µM 5-HT, Φ5-HTmax = 0.38 ± 0.07 pmol/min./oocyte; 1.0 µM 5-HT, Φ5-HTmax

= 3.08 ± 0.08 pmol/min./oocyte; 10 µM 5-HT, Φ5-HTmax = 17.39 ± 0.11

pmol/min./oocyte). Fig. 18A shows normalized data in order to emphasize the 5-HT concentration-dependent shift in apparent cRNA potency (0.1 µM 5-HT, EC50 cRNA = 0.45

± 0.01 ng/oocyte; 1.0 µM 5-HT, EC50 cRNA = 0.84 ± 0.04 ng/oocyte; 10 µM 5-HT, EC50 cRNA

= 2.20 ± 0.12 ng/oocyte; nH = 1.3 for all fits). Over a similar range of 5-HT

concentrations, cRNA potency for I(5-HT) is unchanged (Fig. 18B, 1.0 µM 5-HT, EC50 cRNA

= 3.0 ± 0.4 ng/oocyte; 3.2 µM 5-HT, EC50 cRNA = 3.1 ± 0.4 ng/oocyte; 32 µM 5-HT, EC50 cRNA = 3.5 ± 0.5 ng/oocyte; nH = 1.0 for all fits).

44

10^-7 10^-6 10^-5 10^-4 0

5 10 15 20 25

A B

0.42 ng 21 ng

[5-HT], (M)

I

(5-HT)

(- n A )

10^-7 10^-6 10^-5 10^-4 0

25 50 75 100

[5-HT], (M)

I

(5-HT)

(% M ax .)

Figure 20. 5-HT potency for I5-HT is independent of hSERT expression level.

Oocytes are injected with 0.42 ng/oocyte (open circles) or 21 ng/oocyte (filled circles) hSERT cRNA and cultured for 2 days (24°C). A, I(5-HT) measured at -80 mV, pH 7.6. Data are expressed as the inverse of the actual current. Data represent mean ± SEM from n = 5 or 7 oocytes/condition in a single representative experiment. Lines represent fits to the Hill equation (0.42 ng/oocyte, dashed line, I(5-HT)max = -7.6 ± 0.9 nA, EC50 5-HT = 1.5 ± 0.1 µM, nH = 1.2; 21 ng/oocyte, solid line, I(5-HT)max = – 20.4 ± 0.9 nA, EC50 5-HT = 2.0 ± 0.2, nH = 1.4). B, data from A expressed as a percentage of the respective maximal current.

We also plot Φ5-HT data from the experiment shown in Fig. 18A against [5-HT] to measure changes in 5-HT potency at different levels of cRNA injection (data not

shown). We observe that 5-HT exhibits higher potency at lower hSERT expression (0.42 ng/oocyte, EC50 5-HT = 1.7 ± 0.5 µM; 21 ng/oocyte, EC505-HT = 8.0 ± 4.0 µM; nH = 1.0 for all fits, mean ± SEM from n = 3 separate experiments, data not shown). 5-HT potency is therefore inversely correlated with SERT expression and cRNA potency is inversely correlated with 5-HT concentration.

Expression-level-dependent shifts in substrate potency are also observed in competition assays where we measure concentration-dependent inhibition of Φ5-HT (Fig.

45

-6.0 -5.5 -5.0 -4.5

pK

i

or p E C

50

cRNA injected, (ng/oocyte)

0.1 1 10 100

*

*

Φ_5-HT

I

_(5-HT)

5-HT cocaine

Figure 21. SERT ligands exhibit expression level-sensitive potency shifts for Φ_5-HT but not I(5-HT).

pKi for inhibition of Φ_5-HT by 5-HT (open squares, data from Fig. 19), cocaine (filled triangles, data from Fig. 19), and pEC50 for 5-HT (open circles, data from Fig. 20). pKi values are calculated from Eqn. 4. * p < 0.05 vs. I(5-HT).

19). 5-HT potency for inhibition of Φ5-HT is higher in oocytes injected with 0.42 ng hSERT cRNA than in oocytes injected with 21 ng hSERT cRNA (Fig. 19A). Inhibitory potency for the non-transported ligand cocaine follows the same trend as 5-HT, only the magnitude of the potency shift is larger (Fig. 19B). The transported substrate D- amphetamine (AMPH) shares expression-level sensitivity with 5-HT (0.42 ng/oocyte, IC50 = 40 ± 5.7 µM; 21 ng/oocyte, IC50 = 170 ± 30 µM, data not shown), and the non- transported antidepressant paroxetine (IC50 = 7.9 ± 1.3 nM; 21 ng/oocyte, IC50 = 55.2 ± 9.6 nM, data not shown) shifts similarly to cocaine. pKi values are estimated for 5-HT and cocaine (Eqn. 4, pKi = -5.8 and –5.12 for 5-HT and pKi = –5.73 and –4.7 for cocaine in oocytes injected with 0.42 ng/oocyte and 21ng/oocyte, respectively).

In contrast to Φ5-HT, the potency for 5-HT to elicit I(5-HT) is insensitive to hSERT expression level (Fig. 20). At low hSERT cRNA (0.42 ng/oocyte), 5-HT potency for I(5-

46

HT) is similar to that seen for Φ5-HT (Fig. 20, I(5-HT)max = -7.6 ± 0.9 nA, EC50 5-HT = 1.6 ± 0.9 µM, nH = 1.0, mean ± SEM from n = 5 oocytes). At higher expression level (21

ng/oocyte), 5-HT potency remains essentially unchanged (Fig. 20, I(5-HT)max = –20.4 ± 0.9 nA, EC50 5-HT = 2.4 ± 0.4, nH = 1.0, n = 7 oocytes).

Fig. 21 summarizes changes in ligand potency observed at low and high SERT expression. Inhibitory potency decreases 4 to 5 fold for 5-HT and 10 fold for cocaine when cRNA is increased from 0.42 ng/oocyte to 21 ng/oocyte (Fig. 19). Over the same range of cRNA, however, 5-HT potency for I(5-HT) does not change substantially (Fig.

20). Substrate and inhibitor potency is therefore sensitive to SERT expression level when measured in the functional context of 5-HT transport, but not 5-HT-induced current. The finding that SERT transport and current properties are differentially sensitive to expression level suggests that SERT may interact with a protein or cellular factor that modulates its function. One possibility is that ρ is sensitive to inter-subunit interactions in an oligomeric SERT functional complex ^119,132.

In order to determine whether rSERT and D98G exhibit functional interactions, we inject oocytes with rSERT cRNA alone (8.3 ng/oocyte) or mixed with D98G (rSERT, 8.3 ng + D98G, 33.1 ng/oocyte) and measure Φ5-HT and I(5-HT) during 1 min. (24°C) assays at –80 mV to obtain ρ. Fig. 22A shows that co-injection of rSERT + D98G

attenuates total charge movement, Q(5-HT), by 51% compared to rSERT alone. However, the charge movement associated with 5-HT flux, Q5-HT, is unaffected (Fig. 22B). D98G consequently decreases ρ by 35% compared to rSERT (Fig. 22C). D98G alone mediates no detectable I(5-HT) above that seen in non-injected oocytes (data not shown). D98G therefore selectively attenuates I(5-HT) when co-expressed with rSERT.

The effect of D98G in oocytes is specific, since when we co-inject rSERT with a Shaker K⁺ channel (rSERT, 8.3 ng + ZH4IR, 2.3 ng/oocyte), we observe no effect on Φ5- HTor I(5-HT). However, in the same batch of oocytes we do record a large outward current (up to 16 µA at +100 mV, data not shown). Given that under voltage clamp in oocytes, po ≈ 0.75 and i ≈ 1 pA for this Shaker construct ¹⁵⁶, we calculate that there are

~1.3 x 10⁶ Shaker channels expressed on the plasma membrane of an oocyte generating 47

10 µA current at +60 mV (Eqn. 3).

In order to determine whether SERT and D98G interact to alter ion permeation properties, we examine the voltage dependence of I(5-HT) in oocytes injected with rSERT cRNA alone or together with D98G cRNA. Fig. 23A shows representative raw currents from an oocyte injected with rSERT cRNA alone (8.3 ng/oocyte) recorded over the indicated voltage ramp in the absence and presence of 5-HT (10 µM), pH 5.0. I(5-HT) is defined by subtraction (Fig. 15) and plotted as a function of membrane voltage to generate I(5-HT) (V) in Fig. 23B. I(5-HT)(V) exhibits the inward rectification and

0.0 0.2 0.4 0.6

ratio transport

current

rSERT

rSERT + D98G

**

Q

(5-HT)

(

µ

C)

0.15

0.10

0.05

0.0

Q

5-HT

(

µ

C)

0 1 2 3 4 5

C B

A

*

ρ

Figure 22. ρ is sensitive to co-expression of rSERT with D98G.

Oocytes are injected with rSERT cRNA (8.3 ng/oocyte, open bars) or rSERT + D98G (8.3

ng/oocyte + 33.1 ng/oocyte, filled bars) and cultured for 8 days (18°C). A, total charge movement (Q(5-HT)) obtained from integration of I(5-HT); B, charge movement due to 5-HT itself (Q5-HT) while I(5- HT) is induced; C, ρ, the ratio Q_(5-HT)/Q5-HT. Data represent means ± SEM for n = 6 or n = 5

oocytes/condition in a single representative experiment. * p < 0.05, ** p < 0.001 vs. rSERT alone.

48

exponential dependence on membrane potential that is characteristic of SERTs ^95,98 (Fig.

23B). Extracellular acidification (from pH 7.6 to pH 5.0) increases the magnitude of I(5- HT) (V) (see Fig. 15) but does not change the shape of I(5-HT) (V) (data not shown). In oocytes injected with a mixture of rSERT + D98G (8.3 ng/oocyte + 33.1 ng/oocyte, respectively), I(5-HT) is attenuated at all membrane potentials tested (Fig. 23B). At –80 mV, I(5-HT) is inhibited 89% by D98G (Fig. 23B, rSERT, -44.45 ± 9.47 nA; rSERT + D98G, - 5.12 ± 2.19 nA). We are unable to detect I(5-HT) at any membrane potential in oocytes injected with D98G alone (33.1 ng/oocyte, data not shown). Normalizing the data shows that co-expression of rSERT and D98G does not change the shape of the I(5-HT) (V) despite a marked reduction in current amplitude (Fig. 23C).

49